PROJECT SUMMARY How tissues assume their correct shape and topology is a central biological question with immediate relevance to both embryonic development and human disease. This project will elucidate the fundamental physical mechanisms by which solid cell masses form a central cavity, termed a lumen. Lumen formation occurs repeatedly during both early embryonic development and organogenesis, and defines both a functional ?inside? and ?outside? as well as the physical shape of the associated tissue. Previous work has established many of the genetic and biochemical requirements for epithelial polarity and de novo lumen generation. However, lumen formation is an intrinsically physical process ? the transition from a solid cell mass to a hollow, spherical shell. Due to previous technical limitations, surprisingly little is known about the biophysical mechanisms that drive lumen formation. To address this knowledge gap, this project will combine quantitative biophysical measurements with classical cell biological approaches to determine the fundamental physical mechanisms that drive lumen formation and expansion in Madin Darby Canine Kidney (MDCK) epithelial cells, a standard model in the field. Preliminary data indicate that, contrary to expectation, cells can form multiple initial openings, suggesting that lumen formation occurs over two stages: initiation, defined as the formation of at least one small opening, followed by establishment, the growth and stabilization of a single central lumen. Recent work has revealed that human embryonic stem cell (hESCs) under proper 3D culture conditions form hollow spheres with striking similarities to the proamniotic cavity, a lumen that is essential for the initiation of the body plan. In the second part of this project, I will use the experimental approach refined using the MDCK model system to determine the physical mechanism of lumen formation employed by hESCs. This work will determine if the physical processes that govern lumen formation in these two systems, which reflect distinct biological origins and functions, are fundamentally similar or different. In addition, these latter experiments provide insight into the physical mechanisms that drive early human embryogenesis, which is not otherwise accessible due to unavoidable technical and ethical barriers.